Delta-9-tetrahydrocannabinol (THC, also known as dronabinol) is the main biologically active component in the Cannabis plant which has been approved by the Food and Drug Administration (FDA) for the control of nausea and vomiting associated with chemotherapy and, more recently, for appetite stimulation of AIDS patients suffering from the wasting syndrome. The drug, however, shows other biological activities which lend themselves to possible therapeutic applications, such as in the treatment of glaucoma (1), migraine headaches (2, 3), spasticity (4), anxiety (5), and as an analgesic (4). It is because of these-promising biological activities of THC that marijuana has been brought into a public debate relative to its medicinal value. The balance between medicinal use of a drug and the abuse potential is a delicate balance. One of the main points brought by the medicinal marijuana proponents is the fact that the currently available soft gelatin capsule formulation is very expensive and lacks consistency in its effects. The latter point could be explained based on the fact that oral THC has erratic absorption from the gastrointestinal tract, is subject to the first-pass effect resulting in heavy metabolism with production of high levels of 11-OH-THC, and undesirable side effects. Another THC formulation which is currently under development is a pro-drug consisting of THC hemisuccinate formulated in a suppository base (6). This formulation appears to overcome the problems associated with the oral preparation and has been shown to produce consistent bioavailability in animal studies (7). Preliminary clinical investigations show promise for this formulation (8, 9, 10). It is anticipated that other THC formulations will be forthcoming in light of the current interest in the therapeutic activities of cannabis.
Regardless of which formulation is to be used for THC or a pro-drug thereof, a source for the raw material is critical. The currently-approved capsule formulation is prepared from synthetic THC which is extremely expensive to produce. It is thought that should an economic process be developed for isolation of THC from the natural material (cannabis), then the cost of the raw material could be brought down significantly, making it possible to develop such formulations at a reasonable cost to the public. The consequence of this would be the availability of alternative therapies involving THC (or a prodrug thereof) which would help in suppressing the public outcry for approval of marijuana as a medicine.
Several investigations have been carried out over the years to isolate THC from the plant material, mostly to determine its chemical structure or to investigate the phytochemistry of the plant. In 1942, Wollner, et al., (11) reported the isolation of tetrahydrocannabinol from cannabis extract xe2x80x9cred oilxe2x80x9d. Red oil was prepared by extraction of the plant material with ether, followed by distillation of the concentrated extract at room pressure followed by redistillation under reduced pressure (15-50 mm Hg). The oil was acetylated with acetic anhydride, and the acetylated product was subjected to fractional distillation in vacuo. Six fractions were collected. The head and tail fractions were removed. The remaining four fractions which represent the principal fractions (fractions 2, 3, 4, and 5) were combined and passed over silica gel column in benzene and then passed over activated alumina in carbon tetrachloride solution. The product was hydrolyzed by acid, alkali, or ammonia in alcoholic solution. The authors reported that the deacetylated product has, in each case, a different physiological potency than the acetate. All fractions were not pure compounds.
DeRopp, in 1960 (12), described the isolation of THC from the flowering tops of Cannabis sativa. His method involved adsorption chromatography of the methanolic extract of cannabis followed by partition chromatography on Celite using N,N-dimethyl formamide/cychlohexane mixture and high vacuum distillation. The purity of THC was based on paper chromatographic evidence.
The first isolation of the naturally occurring THC in its pure form was reported by Gaoni and Mechoulam in 1964 (13). THC was isolated from the hexane extract of hashish by repeated column chromatography on florisil and alumina. Further purification was carried out by the preparation of the crystalline 3,5-dinitrophenylurethane of THC followed by mild basic hydrolysis to get the pure THC. The purity of THC was proven by thin layer chromatography (TLC) and spectroscopic analysis (IR and NMR).
Korte, et al., in 1965 (14) reported the isolation of THC from the crude extracts of the female inflorescence of Cannabis sativa indica and Cannibis sativa non indica. The crude extracts were chromatographed over activated alumina in order to remove the coloring impurities like carotinoids, chlorophylls and xanthophylls. All the cannabinolic fractions were combined and concentrated to give a brownish-red oil. The oil was further purified by a countercurrent distribution method to get THC which was proved to be identical with that described by Gaoni and Mechoulam (13).
In 1967, Mechoulam and Gaoni (15) reported the isolation of THC from the acidic fraction of the hexane extract of hashish. The hexane extract of hashish was separated into acidic and neutral fractions. The acidic fraction was chromatographed on florisil or acid washed alumina. The column was eluted with pentane-ether mixtures in a manner of increasing polarities. THC was eluted with 15% ether in pentane. Repeated chromatography was carried out by the preparation of crystalline derivative (3,5-dinitrophenylurethane THC, m.p., 115-116xc2x0 C.) followed by hydrolysis.
In 1972, Verwey and Witte (16) reported a method for the preparation of THC by isolation of THC acid from hashish. The hexane extract was shaken with 2% NaOH solution as well as 2% sodium sulphite in an extraction funnel. The alkaline layer was rendered acidic with H2SO4 (pH less than 2), thus precipitating the cannabinoid acids. The oily layer as well as the oily deposits on the wall were extracted with ether. The acid-base extraction process was repeated. THC was obtained from the impure acids by heating the ether solution containing the acids on a sand bath with a temperature of 300xc2x0 C. The ether being evaporated, the evaporating dish was for a moment kept on the sand bath, in this way causing decarboxylation of THC acid. The THC was cleaned by preparative TLC.
In summary, for isolation of THC and other cannabinoid constituents, generally the alcoholic or the petroleum ether or benzene or hexane extract of the plant is separated into neutral and acidic fractions. These fractions are further purified by repeated column chromatography and countercurrent distribution or a combination of these methods. Various adsorbents have been used in column chromatography, especially silica gel, silicic acid, silicic acid-silver nitrate, florisil, acid washed alumina, and acid washed alumina-silver nitrate. Most of the above-discussed methods were used for the preparation of a small amount of THC and not for large-scale production.
If THC is to be prepared in large-scale (kilogram) quantities, an efficient and economic method is needed. Such a method would require an efficient isolation procedure.
The present invention relates to improvements for the obtaining of THC and THC-acid from Cannabis plant material. Simple, high yielding steps are developed which reduce the cost of preparation of THC several fold over the synthetic route.
The present invention relates to improvements in a process which comprises a process wherein Cannabis plant material is extracted with a non-polar organic solvent to provide an extract containing THC and the extract is subjected to fractional distillation under reduced pressure to provide a distillation fraction (distillate) having a high content of THC. The process further comprises subjecting the extract from the plant material to column chromatography prior to fractional distillation. A still further aspect of the process comprises subjecting the distillate from the fractional distillation to column chromatography. Additionally, the invention includes the use of high pressure liquid chromatography (HPLC) in the purification of the extract from the plant material.
The improvement of the present invention relates to a process in which the THC content of cannabis extract or a distillation residue is increased by treating the extract or residue with polar, water miscible organic solvents in admixture with water to form a precipitate and concentrating the filtrate to give a concentrated extract.
A further improvement is a process of chelating THC acid contained in a cannabis extract containing the acid on alumina, washing off the nonacid components with the moderately polar solvents and eluting the alumina with strong polar solvents to provide the separated THC-acid.
The present invention provides an improvement to a procedure for providing an efficient and economic method for isolating THC from Cannabis plant material. The plant material is extracted with a non-polar organic solvent. Useful solvents include lower alkanes, such as, for example, hexane, heptane or iso-octane. The extract containing THC, after solvent removal, is subjected to fractional distillation at reduced pressure and a second distillate is collected. In one embodiment of the present invention, the first distillate is again subjected to fractional distillation at reduced pressure and a second distillate is collected. The second distillate has a THC content of greater than 90% by wt.
In another embodiment of the invention, which is improved by applicants, the crude extract from the plant material is first subjected to column chromatography. One possible method by which the material can be placed on the column is by mixing the extract residue in an organic solvent with a portion of the column packing material and transferring the dried slurry onto the top of a packed column. Direct application of the extract residue in the initial elution solvent (minimum volume) directly to the top of the packed column is also possible. The column is eluted with an organic solvent in a manner such that the column is eluted with a solvent or a solvent mixture with progressively increasing polarity. The fraction or fractions containing the major portion of THC from the column elution is subjected to fractional distillation at reduced pressure. Distillate is collected in the substantially constant boiling temperature range and this distillate was found to contain greater than 90% by weight THC. THC with purity of greater than 95%, preferably greater than 98% can be obtained by further purification of the distillate from fractional distillation by column chromatography or by normal or reversed phase HPLC.
The column chromatography can be carried out using any known packing material including, for example, silica or alumina for normal phase operation or C18 or C8 bonded phase silica for reversed phase operation. Elution of the normal phase chromatography column is carried out with solvents having an increasing polarity. Non-polar solvents include the lower straight chain and branched chain alkanes, including, for example, pentane, hexane, isooctane and petroleum ether. More polar solvents include various organic ethers, alcohols, esters or ketones, including, for example dialkyl ethers, lower alkyl acetates, lower dialkyl ketones and lower alkanols. Illustrative polar solvents include, for example, acetone, ethylacetate, diethylether and isopropyl alcohol. The ratio of non-polar solvent to polar solvent can vary between 100:0 to 80:20.
Elution chromatography under the reversed phase conditions is carried out with solvents having decreasing polarities. These solvents include water or acidic buffer as the polar portion and lower alkanol (such as methanol, ethanol and isopropanol) or acetonitirle as the less polar portion, in mixtures ranging from 50:50 to 0:100 aqueous to organic. The chromatographic process can also be carried out under HPLC conditions in much the same way as described above under either normal or reversed phase operation using a preparative scale column.
Flash distillation is carried out under reduced pressure, i.e. under vacuum at pressures below 1 mm Hg, preferably close to 0.1 mm Hg.
The concentration of delta-9-THC in the initial cannabis extract is a function of the potency (% THC) of the starting plant material. For example, cannabis plant material with THC content of approximately 3% will produce a hexane extract of approximately 35% THC in the first extract and less than 20% in the second extract which might necessitate keeping the first and second extracts separate for further processing. Cannabis biomass of 4% will produce a first hexane extract of approximately 40% THC and a second extract of slightly over 20% THC, while extracts of 5-7% THC plant material will produce a first hexane extract of 45-55% THC with a second extract of approximately 25% THC.
Processing of cannabis extracts of less than 40% THC (whether it be a first extract of a low potency plant material or the second extract of almost any plant material) would be made much more economic if one could pre-treat such extract in a simple step that would result in increasing the THC content to approximately 40% or more. It has been discovered that treatment of xe2x80x9clow THCxe2x80x9d extracts with one of a selection of polar, water missible solvents (such as, for example, lower alkyl alcohols, dialkyl ketones, such as, for example acetone or methylethyl) or acetonitrile in combination with water in various ratios would result in precipitation of significant amount of residue containing small percentage of THC, leaving behind (in solution) the main bulk of THC. A simple filtration step results in removal of the unwanted residue, and evaporation of the solvent of the filtrate results in a concentrated extract with much lower weight than the starting extract and much higher THC content. The resulting extract could then be processed as usual. Furthermore, the residue left from fractional distillation of cannabis extracts is usually of low THC content. This material could be reprocessed in the same manner as discussed above, making the overall process more economical.
Delta-9-THC (1) exists in the fresh cannabis plant material as its precursor xcex949-THC-acid A (2) almost exclusively. 
During the drying and extraction processes variable amounts of the precursor acid 2 is decarboxylated to 1 with the resulting extracts containing a mixture of 1 and 2, in a ratio that depends on the drying and extraction conditions. Under mild conditions of drying of the plant material (40xc2x0-50xc2x0) and mild temperature of evaporation of the extraction solvent, the main component of extract is the acid precursor 2.
The improvement of this invention is, therefore, directed especially to extracts prepared under conditions which preserve the xcex949-THC-acid A and minimize decarboxylation to xcex949-THC.
Treatment of a solution of an extract with alumina allows the strong binding (chelation) of the acid to the exclusion of other components (neutral cannabinoids and the non-cannabinoid components such as terpenes, hydrocarbons, sterols, etc.). The alumina could then be washed (eluted) with non-polar to moderately polar solvents to remove unwanted components followed by elution of xcex949-THC-acid A using strong solvents such as, for example, methanol with varying amounts of acetic acid.
The eluted acid could then be subjected to fractional distillation to give xcex949-THC in a relatively pure form ( greater than 80% chromatographical purity) with a final chromatographic step to remove minor impurities. Alternatively, the eluted acid could be further purified from other similar cannabinoid acids, with the fractional distillation step used at the end to generate xcex949-THC in a pure form.
The alumina chelation, therefore, offers an alternative clean-up step which has the advantage of providing the THC-acid A in relatively pure form in a simple adsorption (filtration) step. This could be especially useful if one desires the separation of the pure acid A for biological evaluation without losing the ability to generate xcex949-THC from the acid by a simple fractional distillation step.
It is to be noted that all three types of alumina solid supports could be used for this process (basic, neutral, and acidic), although basic alumina is preferred.
It will be understood by those skilled in the art that various modifications and substitutions may be made to the invention as described above without departing from the spirit and scope of the invention. Accordingly, it is understood that the present invention has been described by way of illustration and not limitation.